1. Field of the Invention
The present invention relates to an isolated xylanase gene with mutations and a site-specific mutagenesis method thereof. More particularly, the present invention relates to the site-specific mutagenesis method utilized to mutate a forty-first amino acid or a twenty-first amino acid of a xylanase gene from asparagine to aspartic acid so as to form the xylanase gene with mutations.
2. Description of the Related Art
Generally, most of xylans widely exist in structural polysaccharides of plants. The xylan can naturally function as a protective material for celluloses of plans such that the protective material can be a limitation in processing the natural material of plants. For example, in manufacturing pulps of paper materials, there is a need of using a chloride material as a bleaching agent to bleach the pulp due to the fact that the xylan and lignin adhere to surfaces of the celluloses of the plants. After processing the bleaching procedure, the reacted chloride may produce residual products of chemicals which are toxic and carcinogenic substances. The toxic and carcinogenic substances are persistent and bioaccumulating in the natural environment. This seriously destroys the natural environment and the ecological system.
In the livestock industry, animal feed is widely fed and delivered to animal digestive system. The animal feed naturally contains celluloses and hemicelluloses of plants with which to cover its valuable nutrients. The celluloses and hemicelluloses of plants separate the valuable nutrients from enzyme existing in the animal digestive system. In this manner, the valuable nutrients of the animal feed cannot be reacted with the enzyme, or cannot be absorbed by animal intestines of the digestive system. Accordingly, this affects the growth of animals. If the undigested nutrients are excreted from the animal digestive system, there are pollution sources of the undigested nutrients which cause environmental pollution. Hence, there is a need for removal of the xylan from the celluloses and hemicelluloses of plants.
Generally, there is a conventional xylanase which is separated from a rumen microorganism and can be widely used to eliminate the above problem due to the fact that the xylanase can decompose the xylan. In the papermaking industry, the xylanases can decompose the hemicelluloses existing in the paper pulp such that links between the lignin and the celluloses, and between the lignin and the hemicelluloses. Accordingly, the lignin can be released from the paper pulp in the bleaching process. In the food-processing industry, an oligosaccharide is used not only to discompose the hemicelluloses in fruit juices, but also to be as raw materials of foods. In the livestock industry, the oligosaccharide is added to the animal feed. In this manner, the xylanases of the oligosaccharide can be utilized to decompose the xylan in attempting to aid the valuable nutrients to be absorbed by animal intestines of the digestive system. Accordingly, this results in an increase of the absorbed mount of the valuable nutrients.
The primary problem occurring during use of the conventional xylanases is due to the fact that the xylanases possess a lower degree of durability in the broad pH range. Disadvantageously, there is a limitation of use of the xylanases which cannot be widely used in various industries. By way of example, in the papermaking industry, there is a further need of the xylanases for sustaining a higher degree of base materials. Inevitably, the conventional xylanase separated from the rumen microorganism, with a poor durability in the broad pH range, is unsuitable for use in the papermaking industry.
It is a common practice that a mutation method is utilized to improve a characteristic of enzyme in the art. A conventional mutation method is disclosed in the book by Joshi et al. entitled “Hydrogen Bonding and Catalysis”: “a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase,” J. Mol. Biol. (2000) 299, 255-279. A thirty-fifth amino acid of a xylanase gene of bacillus circulans is mutated from asparagine to aspartic acid for reducing a pKa value of the bacillus circulans so as to enhance its acid-resistibility. However, this conventional mutation method partially enhances the acid-resistibility of the xylanase gene, and cannot effectively broaden the pH range of reaction of the xylanase gene.
As is described in greater detail below, the present invention intends to provide an isolated xylanase gene with mutations and a site-specific mutagenesis method thereof. The site-specific mutagenesis method is processed to mutate a forty-first amino acid or a twenty-first amino acid of a xylanase gene from asparagine to aspartic acid so as to form the xylanase gene with mutations in such a way as to mitigate and overcome the above problem. Advantageously, the isolated xylanase gene of the present invention is successful in enhancing its acid base-resistibility, broadening a pH range of reaction and increasing its reaction activity.